Issues in the culture of the extremely thermophilic methanogen, methanothermus fervidus

Abstract

Basic issues in the culture of the extremely thermophilic archaeon, Methanothermus fervidus, have been investigated, including culture medium formulation, substrate yield and product yield coefficient, growth rate and stoichiometry, and H(2) uptake kinetics. The pH optimum for growth of this organism was estimated at 6.9. Growth medium buffered with PIPES instead of bicarbonate supported both increased growth rate and maximum biomass concentration. Substitution of titanium(III) citrate for the reducing agent sodium sulfide improved culture performance as well. However, independent adjustment of iron and nickel concentrations from 11 to 111 microM, respectively, and carbon dioxide partial pressure from 5 to 20 psia did not impact the culture of M. fervidus significantly. An elemental balance approach was utilized to aid in design of a defined medium to support growth to a target maximum biomass concentration of at least 1.0 g dry wt/L. The growth of this organism was limited by H(2) availability in this reformulated culture medium. The maximum growth rate and biomass concentration achieved in anaerobic vials with the defined medium was 0.16 h(-1) and 0.74 g dry wt/L, respectively. This maximum biomass concentration was a 72% improvement over that obtained with a literature-based defined medium. The Monod parameter, K(s), with H(2) as limiting substrate, was estimated at 1.1 +/- 0.4 psia (55 +/- 20 microM in the broth), based on a H(2) consumption study. Representative values for the substrate yield, Y(X/CO(2) ), and product yield coefficient, Y(CH(4)/) (X), were determined experimentally to be 1.78 +/- 0.04 g dry wt/mol CO(2), and 0.52 +/- 0.01 mol CH(4)/g dry wt, respectively. A bench-scale fermentation system suitable for the culture of extremely thermophilic anaerobes was designed and constructed and proved effective for the culture of M. fervidus.

Sulfide assessment in bioreactors with gas replacement

“…(6). This is consistent with additional studies (not shown) which demonstrated that the decay was similar at pH 5.4 and 5.9.…”

“…One study showed that the substitution of titanium citrate for Na 2 S greatly improved the growth of Methanothermus fervidus and increased the production of methanol [6]. Another study showed that the addition of Na 2 S had no effect on the growth of Clostridium aceticum [7].…”

“…Another study showed that the addition of Na 2 S had no effect on the growth of Clostridium aceticum [7]. Furthermore, the reported optimal sulfide concentrations used in fermentations varied greatly, ranging from 1.3 to 136 mM [1][2][3][4][5][6][7][8][9].…”

Reactor-Scale Cultivation of the Hyperthermophilic Methanarchaeon Methanococcus jannaschii to High Cell Densities

“…Our observation is in contrast to the reported fragility of Methanococcus vannielii (12), which is why this organism has been cultivated in a 400-liter reactor without mechanical mixing. A sensitivity to higher agitation rates has also been seen with Methanothermus fervidus (22), even though the organism's cell envelope compositions (15) suggest a capacity to withstand high shear; a very likely explanation for this observation is the inability of the reactor system in use to prevent oxygen contamination (22). Our observations suggest that other methanogens with seemingly weaker cell envelopes should be tested for shear sensitivity while developing a method for mass culture with sparingly soluble gases as substrates.…”

Anaerobic processes

“…strain CALS-I (Zinder and Anguish, 1992). Selection of media and proper environmental conditions for the cultivation of an extremely thermophilic methanogen, Methanothermousfervidus, was presented by Pepper and Monbouquette ( 1993). Methanogenic bacteria were divided into two groups based on the presence of oxidative or reductive citric acid pathways (Sprott et al, 1993).…”